Restriction with two incompatible enzymes - (Jan/15/2007 )

I need to cut a plasmid at two sites. Unfortunately, the only restriction enzymes I found that cut at those sites are not compatible (buffer or temperature). Is it best if I do the restriction sequentially (first with one enzyme and then the other). What should I watch out for if I do that?

Thanks a lot

Yep, I would do it sequentially.

Look up the buffer composition for both enzymes. You might be able to get away with simply adding more NaCl for the second reaction. You want to be careful that the first enzyme does not exhibit start activity under the second reaction conditions. Otherwise you need to heat inactivate the enzyme or do a DNA purificatio between steps.

I would precipitate the DNA between the 2 digests.

No need to precipitate if one enzyme works like 25% buffer A and 100 % buffer B, while the second enzyme is 100% buffer A and 25% buffer B. There should be no adverse effects of having the two enzyme together for the second reaction in buffer B as long as the glycerol concentration doesn't get too high.